Design of donecopride, a dual serotonin subtype 4 PNAS PLUS receptor agonist/acetylcholinesterase inhibitor with potential interest for Alzheimer’s disease treatment Cédric Lecouteya, Damien Hedoua, Thomas Freretb, Patrizia Giannonic,d,e, Florence Gavenc,d,e, Marc Sincea, Valentine Bouetb, Céline Ballandonnea, Sophie Corvaisiera,b, Aurélie Malzert Fréona, Serge Mignania, Thierry Cresteilf, Michel Boulouardb, Sylvie Claeysenc,d,e, Christophe Rochaisa,1, and Patrick Dallemagnea,1 aUniversité de Caen Basse-Normandie, Centre d’Etudes et de Recherche sur le Médicament de Normandie, F-14032 Caen, France; bUniversité de Caen Basse-Normandie, Groupe Mémoire et Plasticité comportementale, F-14032 Caen, France; cCentre National de la Recherche Scientifique, Unité Mixte de Recherche-5203, Institut de Génomique Fonctionnelle, F-34000 Montpellier, France; dInstitut National de la Santé et de la Recherche Médicale U661, F-34000 Montpellier, France; eUniversités de Montpellier 1 and 2, Unité Mixte de Recherche-5203, F-34000 Montpellier, France; and fInstitut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique Unité Propre de Recherche 2301, F-91198 Gif sur Yvette, France Edited* by Joanna S. Fowler, Brookhaven National Laboratory, Upton, NY, and approved July 23, 2014 (received for review June 5, 2014) RS67333 is a partial serotonin subtype 4 receptor (5-HT4R) agonist in rodents, and it even reverses the deleterious effect of atropine that has been widely studied for its procognitive effect. More re- (18) or scopolamine during this same task. cently, it has been shown that its ability to promote the nonamy- Based on the structural analogy existing between RS67333 and loidogenic cleavage of the precursor of the neurotoxic amyloid-β donepezil (Fig. 1), we postulated that RS67333 could improve α peptide leads to the secretion of the neurotrophic protein sAPP . learning and memory by not only activating 5-HT4R but also, This effect has generated great interest in RS67333 as a potential inhibiting acetylcholinesterase (AChE) activity. Indeed, several treatment for Alzheimer’s disease (AD). We show herein that early studies reported that the inhibition of AChE improved RS67333 is also a submicromolar acetylcholinesterase (AChE) in- cognition and that this effect is the main reason for the initial use hibitor and therefore, could contribute, through this effect, to of donepezil, galantamine, and rivastigmine as cognitive enhancers the restoration of the cholinergic neurotransmission that becomes in AD (19, 20). AChE inhibition has also been reported to im- PHARMACOLOGY altered in AD. We planned to pharmacomodulate RS67333 to en- prove performances in healthy rodents or animal models of hance its AChE inhibitory activity to take advantage of this pleio- memory deficiency (21). The hypothesis of an involvement of tropic pharmacological profile in the design of a novel multitarget- AChE inhibition by RS67333 in memory improvement is an issue directed ligand that is able to exert not only a symptomatic but that has never been tested. also, a disease-modifying effect against AD. These efforts allowed Furthermore, such a pharmacological profile could be also h us to select donecopride as a valuable dual ( )5-HT4R partial ago- exploited to lead to pleiotropic compounds that are theoretically K = h nist ( i 10.4 nM; 48.3% of control agonist response)/( )AChEI useful in AD treatment. Indeed, today, it is well-established that CHEMISTRY (IC = 16 nM) that further promotes sAPPα release (EC = 11.3 50 50 5-HT4R activation not only favors ACh release but also, is in- nM). Donecopride, as a druggable lead, was assessed for its in vivo volved in the nonamyloidogenic cleavage of amyloid precursor procognitive effects (0.1, 0.3, 1, and 3 mg/kg) with an improve- protein (APP) in the neurotrophic sAPPα fragment, with se- ment of memory performances observed at 0.3 and 1 mg/kg on cretion that is detrimental to amyloid-β peptide (Aβ) production the object recognition test. On the basis of these in vitro and in (22–24). However, inhibiting the catalytic activity of AChE is vivo activities, donecopride seems to be a promising drug candi- date for AD treatment. Significance mong the large family of serotonin receptors (5-HTR), some Targeting more than one molecular cause implied in the Aof them, such as the subtype 4 (5-HT4R), are of particular pathogenesis of Alzheimer’s disease (AD) with a sole drug is interest in improving memory performance and therefore, de- considered a promising challenge, because it may address the ’ creasing memory deficits, such as those that occur in Alzheimer s numerous failures that recently occurred during clinical trials disease (AD). In the CNS, they are located in structures that are that were conducted in this area. Donecopride has been primarily involved in cognitive functions, like the olfactory designed by us as a multitarget-directed ligand, targeting both tubercles, basal ganglia, septum, substantia nigra, superior col- serotonin subtype 4 receptor and acetylcholinesterase with liculi, hippocampus, and cortex. Several compounds act as ago- excellent in vitro activities. The latter seems able to not only nists to 5-HT4R (BIMU1, BIMU8, RS17017, SL65.0155, VRX- restore the cholinergic neurotransmission altered in AD but 03011, prucalopride, RS67333, and RS67506). One of the most also, promote the secretion of a neurotrophic protein that is affine (pKi = 7.88) and selective vs. other receptors is RS67333, detrimental to the neurotoxic amyloid-β peptide. With its ex- which acts as a partial agonist (1). With respect to the potential cellent drugability, donecopride further displayed significant therapeutic modulation of 5-HT4R with RS67333 and excluding procognitive effects in mice and generated a promising lead for its putative antidepressant-like activity (2–4), most studies a previously unidentified approach in AD treatment. focused on the promnesic or antiamnesic actions of this com- pound. These effects on cognitive functions that concern learn- Author contributions: T.F., M.B., S. Claeysen, C.R., and P.D. designed research; C.L., D.H., ing and memory are probably, in part, because of the fact that T.F., P.G., F.G., M.S., V.B., C.B., S. Corvaisier, A.M.F., and T.C. performed research; T.F., S.M., M.B., S. Claeysen, C.R., and P.D. analyzed data; and T.F., V.B., S. Claeysen, C.R., and P.D. the pharmacological stimulation of these receptors increases the wrote the paper. release of ACh in the hippocampus and cortex, and it also The authors declare no conflict of interest. – increases serotonin, dopamine, and GABA release (5 13). *This Direct Submission article had a prearranged editor. Concerning the selective aspects of memory functions, RS67333 1 To whom correspondence may be addressed. Email: [email protected] or has been shown to improve object recognition in adult (14, 15) [email protected]. and aged animals (16, 17) and place recognition (10) in rodents. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. It also increases spatial learning on the Morris water maze task 1073/pnas.1410315111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1410315111 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 RS67333 Donepezil Donecopride (MR31147) Fig. 2. Donecopride (MR31147) is issued from RS67333 through a hit-to- Fig. 1. RS67333 and donepezil are chemically close. lead chemistry process. widely used to restore cholinergic neurotransmission in AD, and share common intracyclic nitrogen able to engage a Π-cation interacting with the peripheral anionic site (PAS) of this enzyme interaction with a Phe in the aromatic gorge of AChE (29). could also reduce amyloid aggregation, for which AChE would Conversely, some other 5-HT R agonists belonging to other be responsible (25). These activities seem to be synergistic when 4 structural families are devoid of such activity (Table 1). they are associated in an AD animal model, which we have re- On the basis of these results, RS67333 was chosen as a hit to cently shown in mice (26). A second pharmacological approach perform a hit-to-lead chemistry study. Numerous derivatives of based on the fact that a single compound may be able to hit RS67333 were synthesized. Among them, the compound MR31147 multiple targets is now emerging. This concept, called multi- (donecopride) was selected for its outstanding in vitro activity target-directed ligands (MTDLs) (27, 28), would have inherent toward both (h)5-HT R and (h)AChE. The cyclohexyl moiety, advantages over a combination of drugs called multiple medi- 4e introduced into the structure of donecopride, seems to be a cation therapy. It would specially obviate the problems linked to compromise between the N-butyl group of RS67333 and the the complexity of the pharmacokinetic profile of the combined bulky benzyl group of donepezil and is able to potentiate drugs and the risk of drug–drug interactions. Moreover, MTDL both activities. could also alleviate compliance difficulties associated with mul- tiple medication therapy. It has also been shown that MTDLs Donecopride Is a Potent, Selective, and Partial (h)5-HT R Agonist. generally show a higher synergistic effect than that observed with 4e Donecopride (Fig. 2) is an (h)5-HT4eR ligand that is as potent a combination of drugs. Numerous examples of MTDL against as RS67333 (Table 2). It behaves like a partial agonist, with its AD have been recently described (27). Most of them associate an intrinsic activity of 48% vs. 49% for RS67333 (Fig. 3). This AChE inhibitory effect with another activity hitting another profile is in accordance with our objective, because prolonged molecular target of AD, such as antioxidant effect, monoamine exposure of G protein-coupled receptors to an agonist can result oxidase inhibition, calcium channel blocking effect, metal che- in receptor desensitization, which is often dependent on the de- lating activity, etc.